In situ restoration of apatite-based chromatography resins

ABSTRACT

Methods and compositions are provided for treatment of an apatite-based resin from which retained solutes have been eluted by an elution buffer that contains an alkali metal salt with solutions of calcium ion, phosphate ion, and hydroxide separately from any sample loading and elution buffers. The treatment solutions restore the resin, reversing the deterioration that is caused by the alkali metal salt in the elution buffer.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present patent application claims benefit of priority to U.S.Provisional Patent Application No. 61/653,172, filed May 30, 2012, whichis incorporated by reference for all purposes.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to apatite-based chromatographic resins and theiruse in the purification of proteins and other target molecules frombiological samples.

2. Description of the Prior Art

Apatite in its various forms, examples of which are hydroxyapatite,ceramic hydroxyapatite, fluoroapatite, and fluoride-enhanced apatite, isused as a chromatographic solid phase in the separation and purificationof a wide variety of target molecules by way of binding mechanisms thatinvolve either affinity, ion exchange or hydrophobic interactions, orcombinations of these mechanisms. Apatite-based chromatography resinsare particularly useful in protein purifications, notably purificationsof recombinant proteins from host cell proteins, aggregates, endotoxin,and DNA. Sample loading of an apatite-based resin, particularly withproteins, is most often conducted by first equilibrating the resin to pH6.5 with phosphate buffer at 2 mM to 5 mM, and then loading the samplein a solution at the same pH and buffer content. Once the sample isloaded, unbound components are washed from the resin and the targetmolecule is eluted with an elution buffer that typically contains analkali metal salt, most often at pH of 8.0 or below. The equilibrationand loading buffers both saturate the hydroxyapatite surface withhydroxonium ions (H3O+), which are then desorbed upon exposure to theelution buffer. This desorption causes the resin to deteriorate overtime, resulting in a loss of resin mass and a decline in the particlestrength of the resin. Of potential relevance to this issue is thedisclosure of commonly owned, co-pending U.S. patent application Ser.No. 13/205,354, filed Aug. 8, 2011, entitled “Elution of Proteins FromHydroxyapatite Resins Without Resin Deterioration,” inventor L. J.Cummings. That disclosure describes the use of elution buffers thatinclude calcium ions and phosphate ions at a pH of 6.0 or below, therebyboth eluting the protein and treating the resin with these ions at thesame time. While this buffer is effective in inhibiting the resindeterioration that occurs without the inclusion of the calcium ions inthe buffer, the exposure of the resin to the calcium ions in this methodis limited to the duration of the elution step and the volume of theelution buffer, and the product eluate, although lacking the impuritiesin the original sample, contains calcium ions.

SUMMARY OF THE INVENTION

It has now been discovered that the deterioration of an apatite-basedresin during a chromatographic procedure for purifying a target moleculefrom a sample can be reversed by a post-elution treatment of the resinwith a succession of treatment solutions that contain calcium ion,phosphate ion, and hydroxide ion, by applying these solutions separatelyfrom the sample purification steps such as column equilibration, sampleloading, and elution. The calcium ion, phosphate ion, and hydroxide ionare referred to herein as restoration materials. The treatment solutionscontaining these ions thus do not interfere with the equilibration,loading, and elution buffers, and the ions themselves, referred toherein as “restoration materials,” are not transferred from thesetreatment solutions to the solution of purified target molecule, i.e.,the product solution. In certain cases, phosphate ion may be included inthe equilibration, loading, and elution solutions as part of the buffersin these solutions, and will thus be retained from these solutions inthe purified product. It is demonstrated herein, however, that resinrestoration, whether partial or complete, can thus be performedseparately from product purification, by simply applying the restorationmaterials as additional steps in a protocol consisting of a series ofsolutions passed through the resin. In certain implementations of theinvention, a single resin is used for multiple purifications, i.e.,purifications of target molecules from multiple samples, in succession,with each sample being followed by treatment of the resin with the threerestoration ions which are then cleared from the resin, or at least thecalcium ion is cleared, before loading the succeeding sample. Samplepurification can thus be alternated with column restoration by simplyexchanging the solution being passed through the column while obtainingthe purified protein separate from, i.e., in a solution that is devoidof, the restoration materials.

These and other objects, features, embodiments, and advantages of theinvention will be apparent from the description that follows.

DETAILED DESCRIPTION OF SELECTED EMBODIMENTS

In some embodiments, post-elution treatment of apatite resin with atreatment solution containing calcium ion, a treatment solutioncontaining phosphate ion, and a treatment solution containing hydroxideion, can restore the apatite resin. The use of one or more of thesetreatment solutions to restore apatite resin can be performed aftersteps of equilibration, loading, washing, and elution have beenperformed as described herein.

Calcium ion for use as a restoration material in the proceduresdescribed herein can be supplied by any soluble calcium salt, typicallya salt that is soluble in water, or by calcium hydroxide. Calciumhalides and calcium nitrate are examples of calcium salts that can beused, and calcium chloride is particularly convenient when the alkalimetal halide in the elution buffer is an alkali metal chloride. Thecalcium ion concentration and the amount of the calcium ion solutionpassed through the resin can vary, but will generally be selected as anyamount that will at least partially reverse the deterioration of theresin caused by the alkali metal halide in the elution buffer. Incertain embodiments of the concepts herein, best results will beachieved with a calcium ion concentration of from about 10 ppm (0.25 mM)to about 2000 ppm (49.9 mM), and in many cases about 30 ppm (0.75 mM) toabout 1000 ppm (24.95 mM), including 40 ppm, 50 ppm, 60 ppm, 70 ppm, 100ppm, 150 ppm, 200 ppm, 250 ppm, 500 ppm, or 750 ppm. The volume of thesolution needed to achieve the restoration can vary with the calcium ionconcentration, but in most cases best results will be achieved with fromabout 1.0 to about 10.0 resin volumes of solution, and in many casesfrom about 1.5 to about 6 resin volumes, including 2, 3, 4, or 5 resinvolumes. The calcium ion solution can simply be an aqueous solution of acalcium salt without other solutes, and the pH during treatment withthis solution can be unchanged from the pH of the solution immediatelypreceding this solution.

Phosphate ion for use in the elution buffers can likewise be suppliedfrom any soluble phosphate salt, typically a salt that is soluble inwater. Alkali metal or alkaline earth metal phosphates are examples,with sodium phosphate as a particularly convenient example. As in thecase of the calcium ion, the concentration and amount of the phosphateion solution can vary, but will generally be selected as any amount thatwill at least partially reverse the resin deterioration, particularly incombination with the calcium ion treatment. Here again, the volume ofthe phosphate solution needed to achieve the desired degree ofrestoration will vary with the concentration of the phosphate ion andthe degree of restoration sought to be achieved. In certain embodimentsof the concept herein, best results will be achieved with a phosphateion concentration of from about 50 mM to about 1 M, and in many casesfrom about 200 mM to about 750 mM, including 250 mM, 300 mM, 350 mM, 400mM, 500 mM, 600 mM, or 700 mM. The volume for best results in many caseswill be within the range of about 1.0 to about 20.0 resin volumes, andoften from about 1.5 to about 10.0 resin volumes, including 2, 4, 5, 6,7, 8, or 9 resin volumes. The pH of the phosphate ion solution willgenerally be about 6.5 or above, in many cases from about 6.5 to about9.0, and often most conveniently from about 6.5 to about 7.5, including6.6, 6.8, 7.0, 7.2, and 7.4. A phosphate buffer can thus be used forsupplying the phosphate ion, and the treatment solution in manyembodiments can contain the phosphate buffer, adjusted to the desiredpH, and no other solutes.

A degree of resin restoration can be achieved with either the calciumion treatment preceding the phosphate ion treatment (i.e., the calciumion treatment directly following the application of the alkali metalhalide-containing elution buffer to the resin for elution of the targetmolecule), or with the phosphate ion treatment preceding the calcium iontreatment. In some cases, however, a greater degree of restoration canbe achieved by applying the calcium ion treatment first, followed by thephosphate ion treatment. In some cases as well, best results will beachieved when the resin is treated with a wash solution between theindividual restoration ion treatments to remove any excess calcium,phosphate, or hydroxide ions. A water wash will generally suffice, andthe amounts can vary widely. A typical water wash will be at least about0.2 resin volumes, and in most cases from about 0.2 to about 1.5 or fromabout 0.2 to about 2 resin volumes.

Regardless of whether the calcium ion precedes or follows the phosphateion, the hydroxide ion treatment is applied as the last treatment stepof the resin restoration. Any soluble form of hydroxide ion can be used,preferably water-soluble, and alkali metal hydroxides, and in many casessodium hydroxide, are particularly convenient. As in the cases of thecalcium ion and the phosphate ion, the concentration and quantity ofhydroxide ion solution can vary. The hydroxide ion can clean the resinof residual proteins and contaminants and can also serve as a first steptoward equilibration of the resin to the conditions to be used for thesubsequent sample loading and elution of the target molecule, or forequilibration into conditions suitable for storage. In most cases, bestresults will be achieved with a hydroxide ion concentration of fromabout 0.1 M to about 5.0 M, and in many cases from about 0.3 M to about3.0 M, including 0.5 M, 0.75 M, 1.0 M, 1.25 M, 1.5 M, 2.0 M, or 2.5 M.Suitable volumes of hydroxide ion containing treatment solution rangefrom about 1.0 to about 20.0 resin volumes, and in many cases from about1.5 to about 10.0 resin volumes, including 2, 3, 4, 5, 6, 7, 8, or 9volumes.

The term “apatite-based chromatography resin” as used herein refers toapatite, hydroxyapatite, or any derivatized form of apatite.Fluoroapatite or fluoride-enhanced apatite, for example, can be formedby the reaction of calcium phosphate with ammonium fluoride.Hydroxyapatite is a form of apatite that is available in a variety offorms. Examples are gels such as Bio-Gel HT gel (hydroxyapatitesuspended in sodium phosphate buffer), Bio-Gel HTP gel (a dried form ofBio-Gel HT), and DNA-grade Bio-Gel HTP (a dried form of Bio-Gel HT witha smaller particle size than Bio-Gel HTP). Ceramic hydroxyapatite (CHT)is a chemically pure form of hydroxyapatite that has been sintered athigh temperatures. Ceramic hydroxyapatite is spherical in shape, withparticle diameters ranging from about 10 microns to about 100 microns,and is typically available at nominal diameters of 20 microns, 40microns, and 80 microns. Ceramic hydroxyapatite is macroporous, and isavailable in two types: Type I, with a medium porosity and a relativelyhigh binding capacity, and Type II, with a larger porosity and a lowerbinding capacity. Either porosity can be used, and the optimal porosityfor any particular protein separation or purification will vary with theproteins or the composition of the source mixture. All of theapatite-based resins in this paragraph are available from Bio-RadLaboratories, Inc. (Hercules, Calif., USA). The resin can be used as achromatographic solid phase in the form of a packed bed, and canconstitute either the entire packed bed or a major portion, such as 50%or more by volume, of the packed bed. The packed bed can be retained ina vessel of any configuration, and both the purification performed inthe resin and the restoration of the resin can be performed either as abatch process, a continuous process, or a hybrid batch/continuousprocess. Suitable vessels include columns of extended length relative towidth, and suitable processes include continuous processes such as acontinuous flow through a column.

Deterioration of a resin that occurs upon use can cause the resinparticles to lose their strength and thus to break apart into smallerparticles causing blockage in the column. The deterioration can alsooccur as a chemical breakdown of the apatite, causing a loss of masswhich can in turn result in a loss of column volume, an increase inparticle breakage, or both. All such effects can be reversed by thepresent invention. The reversal of deterioration that can be achieved bythe practice of the present invention can result in a lower rate ofresin mass loss, a lower rate of decline in particle strength, or both.In many cases, the reversal of deterioration can be accompanied byincreases in resin mass, particle strength, or both.

Resin equilibration, sample loading, and elution buffers are well knownin the art, and selection of the optimum buffer in each case will varywith the target molecule being purified and the type of interaction bywhich the target molecule binds to the resin. Of particular interestamong target molecules are proteins, including acidic proteins,antibodies, and monoclonal antibodies, as well as protein fragments, andpolypeptides. Much, if not most, of the resin deterioration in purifyingthese molecules is attributable to the presence of an alkali metalhalide in the elution buffer. In cases where the interaction between thetarget molecule and the resin is one of cation exchange, a highconcentration of alkali metal halide is included in the elution buffer,typically at about 30 mM or higher, and often in the range of about 30mM to about 200 mM, or from about 30 mM to about 2M. In cases where theinteraction is one involving the formation of a calcium coordinationcomplex such as by chelation chemistry, an elution buffer with a lowersodium chloride concentration can be used. The pH of the elution bufferin most cases will be about 8.0 or below, and particularly from about6.0 to about 8.0. In all cases in which the elution buffer contains analkali metal halide, however, the present invention will be beneficial.

The elution buffer can often contain one or more common buffer materialsto maintain a desired pH. Examples are glycine, lysine, arginine,histidine, 2-(N-morpholino)-ethanesulfonic acid (MES),N-(2-acetamido)-2-aminoethanesulfonic acid (ACES),piperazine-N,N′-2-ethanesulfonic acid (PIPES),2-(N-morpholino)-2-hydroxy-propanesulfonic acid (MOPSO),N,N-bis-(hydroxyethyl)-2-aminoethanesulfonic acid (BES),3-(N-morpholino)-propanesulfonic acid (MOPS),N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid (HEPES),3-(N-tris-(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid(TAPSO), 3-(N,N-bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid(DIPSO), N-(2-hydroxyethyl)piperazine-N′-(2-hydroxypropanesulfonic acid)(HEPPSO), 4-(2-hydroxyethyl)-1-piperazine propanesulfonic acid (EPPS),N-[tris(hydroxymethyl)-methyl]glycine (Tricine),N,N-bis(2-hydroxyethyl)glycine (Bicine),[(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]-1-propanesulfonic acid(TAPS), N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonicacid (AMPSO), tris(hydroxymethyl)amino

methane (Tris), and bis[2-hydroxyethyl]iminotris4hydroxymethyl]methane(Bis-Tris). Other buffer materials known in the art may be used as well.

Resin restoration of an apatite-based in accordance with thedescriptions herein can be performed after a single sample purificationon the resin, or after a succession of purifications that collectivelycause greater degradation the resin than a single purification. Incertain implementations of the invention, a single resin is used forrunning a succession of samples on the resin with the restorationtreatment performed after each sample. The number of samples run on theresin in such a procedure may be ten or more, and in many cases twentyor more and even fifty or more. Expressed in ranges, the number ofsamples (each followed by a restoration treatment) can be from ten tothree hundred, from twenty to two hundred, or fifty to one hundred.Resin strength can be observed, for example, by uniaxial confined bulkcompression.

In some cases, restoration treatment can be performed multiple times toachieve enhanced restoration of the apatite resin. For example, adegraded apatite resin may be restored by applying a solution ofphosphate ion, a solution of calcium ion, and a solution of hydroxideion, in succession and in order 2, 3, 4, or 5 times. Alternatively, adegraded apatite resin may be restored by applying a solution of calciumion, a solution of phosphate ion, and a solution of hydroxide ion insuccession and in order 2, 3, 4, or 5 times.

Target molecule purification, and chromatography in general, on anapatite-based resin is typically performed in a sequence of steps thatare known in the art. Prior to equilibration and chromatography, theresin can be pre-equilibrated in a solution such as a salt and/or buffersolution, to displace a solution used for regenerating the resin, or asolution in which the resin is stored, or a solution of the restorationmaterials described above. The composition of the optimalpre-equilibration solution can vary with the composition of the solutionthat is being displaced and the composition of the target molecule, aswell as the sample loading and elution solutions to be used in thepurification. An appropriate pre-equilibration solution can thus includethe same buffer or salt used for performing the chromatography,optionally at a higher concentration than that used in thechromatography. For example, if the solution used to performchromatography comprises sodium phosphate at between about 0.5 mM andabout 50 mM, pre-equilibration may occur in a solution comprising sodiumphosphate at concentrations between about 0.2 M and about 0.5 M, morepreferably in concentrations of sodium phosphate between about 0.3 M andabout 0.4 M, inclusive.

Before the sample is applied to the column, the apatite-based resin isoften equilibrated in the buffer or salt used to load the sample. Any ofa variety of buffers or salts can be used, including those with cationssuch as sodium, potassium, ammonium, magnesium, and calcium, and anionssuch as chloride, fluoride, acetate, phosphate, and citrate. The pH ofthe equilibration solution is typically about 5.5 or higher, in manycases the pH is within the range of about 6.0 to about 8.6 or a range ofabout 6.5 to about 7.5. In some embodiments, equilibration may takeplace in a solution comprising a Tris or a sodium phosphate buffer. Thesodium phosphate buffer may be present at a concentration between about0.5 mM and about 50 mM, or between about 15 mM and 35 mM.

As noted above, all chromatographic, pre-treatment, and post-treatmentsteps described herein can be performed in a conventional chromatographycolumn. The column can be run with or without pressure and from top tobottom or bottom to top, and the direction of the flow of fluid in thecolumn can be reversed during the process. In some cases, it can beadvantageous to reverse the flow of liquid while maintaining the packedconfiguration of the packed bed. The various steps may also be performedin a batch-wise manner in which the resin is first contacted with, thenseparated from, the solutions or liquids used to load the sample, washthe column, and elute the sample. Contacting and separation can beperformed by any suitable means, including gravity, centrifugation, orfiltration.

For protein separations, one example of a class of proteins are thoseproduced by living host cells that have been genetically engineered toproduce the protein. Methods of genetically engineering cells to produceproteins are well known in the art. See, e.g., Ausabel et al., eds.(1990), Current Protocols in Molecular Biology (Wiley, N.Y.). Suchmethods include introducing nucleic acids that encode and allowexpression of the protein into living host cells. The host cells can bebacterial cells, fungal cells, or, preferably, animal cells grown inculture. Examples of bacterial host cells are Escherichia coli cells.Examples of suitable E. coli strains are HB101, DH5α, GM2929, JM109,KW251, NM538, and NM539. In some cases, an E. coli strain that fails tocleave foreign DNA can be used as a host cell. Examples of fungal hostcells are Saccharomyces cerevisiae, Pichia pastoris and Aspergilluscells. Examples of animal cell lines are CHO, VERO, BHK, HeLa, Cos,MDCK, 293, 3T3, and WI38. New animal cell lines can be established usingmethods well known by those skilled in the art, such as bytransformation, viral infection, and/or selection. In some cases, theseparation can be performed on a protein secreted by the host cells. Forexample, the protein can be secreted into culture media and the culturemedia loaded onto the resin.

Further examples of proteins that can be purified in the apatite-basedresins are recombinant fusion proteins comprising one or more constantantibody immunoglobulin domains, optionally an FC portion of anantibody, and a protein identical to or substantially similar to one ofthe following proteins: an flt3 ligand, a CD40 ligand, erythropoeitin,thrombopoeitin, calcitonin, Fas ligand, ligand for receptor activator ofNF-kappa B (RANKL), tumor necrosis factor (TNF), TNF-relatedapoptosis-inducing ligand (TRAIL), thymic stroma-derived lymphopoietin,granulocyte colony stimulating factor, granulocyte-macrophage colonystimulating factor (GM-CSF), mast cell growth factor, stem cell growthfactor, epidermal growth factor, RANTES, growth hormone, insulin,insulinotropin, insulin-like growth factors, parathyroid hormone,interferons, nerve growth factors, glucagon, interleukins 1 through 18,colony stimulating factors, lymphotoxin-β, leukemia inhibitory factor,oncostatin-M, and various ligands for cell surface molecules ELK and Hek(such as the ligands for eph-related kinases or LERKS).

Still further examples are recombinant fusion proteins containing one ormore constant antibody immunoglobulin domains, optionally an FC portionof an antibody, plus a receptor for any of the above-mentioned proteinsor proteins substantially similar to such receptors. These receptorsinclude both forms of TNFR (referred to as p55 and p75), Interleukin-1receptors types I and II, Interleukin-2 receptor, Interleukin-4receptor, Interleukin-15 receptor, Interleukin-17 receptor,Interleukin-18 receptor, granulocyte-macrophage colony stimulatingfactor receptor, granulocyte colony stimulating factor receptor,receptors for oncostatin-M and leukemia inhibitory factor, receptoractivator of NF-kappa B (RANK), receptors for TRAIL (including TRAILreceptors 1, 2, 3, and 4), and receptors that comprise death domains,such as Fas or Apoptosis-Inducing Receptor (AIR).

Other examples of proteins are differentiation antigens (referred to asCD proteins) and their ligands, which are fused to at least one constantantibody immunoglobulin domain, optionally an FC portion of an antibody.Such antigens are disclosed in Leukocyte Typing VI (Proceedings of theVIth International Workshop and Conference, Kishimoto, Kikutani, et al.,eds., Kobe, Japan, 1996). Examples of such antigens are CD27, CD30,CD39, CD40, and ligands thereto (CD27 ligand, CD30 ligand, etc.).

Still further examples are enzymatically active proteins and theirligands. Examples are recombinant fusion proteins comprising at leastone constant antibody immunoglobulin domain plus all or part of one ofthe following proteins or their ligands or a protein substantiallysimilar to one of these: metalloproteinase-disintegrin family members,various kinases, glucocerebrosidase, superoxide dismutase, tissueplasminogen activator, Factor VIII, Factor IX, apolipoprotein E,apolipoprotein A-I, globins, an IL-2 antagonist, alpha-1 antitrypsin,TNF-alpha Converting Enzyme, ligands for any of the above-mentionedenzymes, and numerous other enzymes and their ligands.

Still further examples are antibodies or portions thereof and chimericantibodies, i.e., antibodies having at least one human constant antibodyimmunoglobulin domain coupled to one or more murine variable antibodyimmunoglobulin domains, or fragments thereof, and conjugates of anantibody and a cytotoxic or luminescent substance. Such substancesinclude: maytansine derivatives (such as DM1); enterotoxins (such as aStaphlyococcal enterotoxin); iodine isotopes (such as iodine-125);technium isotopes (such as Tc-99m); cyanine fluorochromes (such asCy5.5.18); and ribosome-inactivating proteins (such as bouganin,gelonin, or saporin-S6). Examples of antibodies or antibody/cytotoxin orantibody/luminophore conjugates are those that recognize any one orcombination of the above-described proteins and/or the followingantigens: CD2, CD3, CD4, CD8, CD11a, CD14, CD18, CD20, CD22, CD23, CD25,CD33, CD40, CD44, CD52, CD80 (B7.1), CD86 (B7.2), CD147, IL-1α, IL-1β,IL4, IL-5, IL-8, IL-10, IL-2 receptor, IL-4 receptor, IL-6 receptor,IL-13 receptor, IL-18 receptor subunits, PDGF-β, VEGF, TGF, TGF-β2,TGF-β1, EGF receptor, VEGF receptor, C5 complement, IgE, tumor antigenCA125, tumor antigen MUCI, PEM antigen, LCG (which is a gene productthat is expressed in association with lung cancer), HER-2, atumor-associated glycoprotein TAG-72, the SK-1 antigen, tumor-associatedepitopes that are present in elevated levels in the sera of patientswith colon and/or pancreatic cancer, cancer-associated epitopes orproteins expressed on breast, colon, squamous cell, prostate,pancreatic, lung, and/or kidney cancer cells and/or on melanoma, glioma,or neuroblastoma cells, the necrotic core of a tumor, integrin alpha 4beta 7, the integrin VLA-4, B2 integrins, TRAIL receptors 1, 2, 3, and4, RANK, RANK ligand, TNFα, the adhesion molecule VAP-1, epithelial celladhesion molecule (EpCAM), intercellular adhesion molecule-3 (ICAM-3),leukointegrin adhesin, the platelet glycoprotein gp Ilb/IIIa, cardiacmyosin heavy chain, parathyroid hormone, rNAPc2 (which is an inhibitorof factor VIIa-tissue factor), MHC I, carcinoembryonic antigen (CEA),alpha-fetoprotein (AFP), tumor necrosis factor (TNF), CTLA-4 (which is acytotoxic T lymphocyte-associated antigen), Fc-γ-1 receptor, HLA-DR 10β,HLA-DR antigen, L-selectin, IFN-.gamma., Respiratory Syncitial Virus,human immunodeficiency virus (HIV), hepatitis B virus (HBV),Streptococcus mutans, or Staphlycoccus aureus.

EXAMPLE 1 Control Experiment

This example illustrates the deterioration of a hydroxyapatite resinover a series of cycles exposing the resin to conditions that simulatethose encountered in protein separations (but without loading andeluting protein). The experiment was performed on a column measuring 20cm in length and 2.2 cm in internal diameter, with an internal volume of76 mL, and the packing was ceramic hydroxyapatite Type I in 40-micronparticles weighing approximately 48 grams, the resulting mobile phaseflow rate through the column being 250 cm/h. A series of 25 consecutivecycles were performed, each cycle consisting of the following eightsteps:

TABLE I Treatment Protocol for Simulated Cycles of Separation and ColumnRestoration Amount Column Volume Time in Step Mobile Phase Volumes in mLminutes 1 Water 1.0 76.0 4.8 2 400 mM NaPi, pH 7.0 3.0 228.1 14.4 3 10mM NaPi, pH 6.8 6.0 456.2 28.8 4 10 mM NaPi, 1.0M NaCl, pH 6.5 6.0 456.228.8 5 Water 2.0 152.1 9.6 6 400 mM NaPi, pH 7.0 2.0 152.1 9.6 7 Water1.0 76.0 4.8 8 1M NaOH 2.0 152.1 9.6

In this protocol, Step 2 is a conditioning step to lower the pH of thecolumn following the alkali treatment of Step 8; and Steps 3 and 4expose the column to the conditions that are generally present duringcolumn equilibration, sample loading, and elution. Measurements ofparticle mass and particle strength (by uniaxial confined bulkcompression, “UCBC”) were taken before the first cycle and after thelast cycle in each of three segments of the column—the top 25%, themiddle 50%, and the bottom 25% (the mobile phase entry being at the topof the column). The results are listed in Table II below.

TABLE II Changes in Solid Phase Mass and Strength at Three ColumnLocations for Simulated Cycles of Separation and Column RestorationResin Mass Particle Strength Packing After (UCBC @ 6.00 mm) Loca- 25Percent Percent tion Start Cycles Change N psi Change Top 12.00 g 11.11g −7.42% 136.9 516 −49% 25% Middle 23.90 g 22.26 g −6.86% 156.6 590 −41%50% Bottom 12.00 g 11.23 g −6.42% 132.05 498 −50% 25% Total 47.90 g44.60 g −6.89% Control: 266.4 1003

The data in Table II indicate that the resin experienced chemicalmodification as evidenced by a loss of mass and a decline in particlestrength. The overall mass loss was 6.89% and was approximately uniformthroughout the height of the column. The decline in particle strengthwas greatest at the top and bottom, but generally extended throughoutthe column as well.

EXAMPLE 2

This example illustrates the result of including column restoration inaccordance with the present invention in the column cycling ofExample 1. A series of runs identical to those of Example I wereperformed on an identical hydroxyapatite column, except that a calciumchloride solution was passed through the column after the solutions thatsimulated the conditions for equilibration, sample loading, washing, andelution. Each cycle thus consisted of the following nine steps:

TABLE III Treatment Protocol for Simulated Cycles of Separation andColumn Restoration Amount Column Volume Time in Step Mobile PhaseVolumes in mL minutes 1 Water 1.0 76.0 4.8 2 400 mM NaPi, pH 7.0 3.0228.1 14.4 3 10 mM NaPi, pH 6.8 6.0 456.2 28.8 4 10 mM NaPi, 1.0M NaCl,pH 6.5 6.0 456.2 28.8 5 50 mM CaCl₂•2H₂O 3.0 228.1 14.4 6 Water 2.0152.1 9.6 7 400 mM NaPi, pH 7.0 2.0 152.1 9.6 8 Water 1.0 76.0 4.8 9 1MNaOH 2.0 152.1 9.6

As in Example 1, 25 cycles were performed with measurements of particlemass and particle strength taken before the first cycle and after thelast cycle. The measurements indicate that particle mass for the entirecolumn increased by 4.72% and particle strength increased by 9%, overthe course of the 25 cycles.

EXAMPLE 3

This example is a further illustration of column restoration inaccordance with the present invention, illustrating the changesoccurring in different parts of the same column to provide a directcomparison with the Control Example (Example 1). The columnconfiguration was identical to that of the preceding examples, thetreatment protocol was identical to that of Table III, and changes inmass and particle strength were obtained for each of three segments ofthe column before the first cycle and after the last cycle. The resultsare shown in Table IV below.

TABLE IV Changes in Solid Phase Mass and Strength at Three ColumnLocations for Simulated Cycles of Separation and Column RestorationParticle Strength Resin Mass (UCBC @ 6.00 mm) Packing After 25 PercentPercent Location Start Cycles Change N psi Change Top 25% 12.00 g 11.89g −0.92% 171.4 646 −23% Middle 23.90 g 25.07 g +4.90% 209.3 789 −6% 50%Bottom 12.00 g 12.94 g +7.83% 251.4 947 +13% 25% Total 47.90 g 49.90 g+4.18% Control: 222.4 838

Table IV shows that a slight loss in mass occurred in the upper 25% ofthe column while gains in mass occurred in the middle 50% and lower 25%of the column. The column as a whole increased in mass by 4.18%.Particle strength declined in the upper 25%, remained approximatelyneutral in the middle 50%, and increased by 13% in the lower 25%.Compare these results to those of Example 1 (Table II), where the masschange was a loss of 6.89%, and the particle strength declined in eachof the three column segments, with a 50% decline in the lower 25%.

EXAMPLE 4

This example illustrates the effect of treating a column with a seriesof restoration treatments in accordance with the present invention butwithout the intervening treatments that simulate the conditionsencountered during column equilibration, sample loading, washing, andelution. The column configuration was identical to those of thepreceding examples, but each of the 25 cycles utilized the treatmentprotocol shown in Table V below.

TABLE V Treatment Protocol for Column Restoration Amount Column VolumeTime in Step Mobile Phase Volumes in mL minutes 1 Water 1.0 76.0 4.8 2400 mM NaPi, pH 7.0 3.0 228.1 14.4 3 Water 6.0 456.2 28.8 4 50 mMCaCl₂•2H₂O 6.0 456.2 28.8

Measurements of resin mass and particle strength were taken in threesegments of the column as in Example 3, both before the first cycle andafter the 25th cycle, and the results are shown in Table VI.

TABLE VI Changes in Solid Phase Mass and Strength at Three ColumnLocations Over 25 Cycles of Column Restoration Particle Strength ResinMass (UCBC @ 6.00 mm) Packing After 25 Percent Percent Location StartCycles Change N psi Change Top 25% 12.00 g 11.75 g −2.08% 200.5 755 −25%Middle 23.90 g 23.66 g −1.00% 277.5 1046 +4% 50% Bottom 12.00 g 11.90 g−0.83% 287.6 1084 +8% 25% Total 47.90 g 47.31 g −1.23% Control: 266.31003

The small values of percent change in mass indicate that very little, ifany, structural modification of the resin occurred. The 25% decrease inparticle strength in the upper 25% of the column is characteristic ofpacked columns of apatite that have been used for repeated proteinpurifications without including calcium chloride in the equilibration,load, and elution solutions. The increase in the middle and lowersections show the positive effects of the calcium restoration material.

EXAMPLE 5

This example illustrates the effect of treating a column with a seriesof restoration treatments in accordance with the present invention afterpartial exposure to intervening treatments that simulate the conditionsencountered during sample purification, except without exposure toalkali metal that would otherwise occur during elution or equilibration.The column configuration was identical to those of the precedingexamples, but each of the 25 cycles utilized the treatment protocolshown in Table VII below.

TABLE VII Treatment Protocol for Simulated Cycles of Separation andColumn Restoration But Without Alkali Metal Halide Exposure AmountColumn Volume Time in Step Mobile Phase Volumes in mL minutes 1 Water2.0 76.0 9.6 2 50 mM CaCl₂•2H₂O 3.0 228.1 14.4 3 Water 1.0 76.0 4.8 4400 mM NaPi, pH 7.0 3.0 228.1 14.4 5 1M NaOH 2.0 152.1 9.6 6 Water 1.0152.1 4.8 7 400 mM NaPi, pH 7.0 2.0 152.1 9.6

Measurements of resin mass and particle strength were taken in threesegments of the column as in Example 3, both before the first cycle andafter the 25th cycle, and the results are shown in Table VIII.

TABLE VIII Changes in Solid Phase Mass and Strength at Three ColumnLocations Over 25 Cycles of Column Restoration Particle Strength ResinMass (UCBC @ 6.00 mm) Packing After 25 Percent Percent Location StartCycles Change N psi Change Top 25% 12.00 g 11.77 g −1.92% 168.8 636 −37%Middle 23.90 g 23.87 g −0.13% 263.6 993 −1% 50% Bottom 12.00 g 12.28 g+2.33% 235.4 887 −12% 25% Total 47.90 g 47.92 g +0.04% Control: 266.31003

As in Example 4, the small values of percent change in mass indicatedthat very little, if any, structural modification of the resin occurred,and that a loss in particle strength occurred in the upper 25% of thecolumn as is typical of packed columns of apatite that have been usedfor repeated protein purifications. The lesser losses in the middle andlower sections (and the increase in mass in the lower section) show thepositive effects of the restoration protocol of the present invention.

EXAMPLE 6

This example illustrates a variation on the protocols of the precedingexamples, by reversing the order of the calcium and phosphate ions.Otherwise, the treatment protocol for each of the 25 cycles was similarto those of Table III (Example 2) above. The column configuration wasidentical to those of the preceding examples and each cycle consisted ofthe following eleven steps:

TABLE IX Treatment Protocol for Simulated Cycles of Separation andColumn Restoration Amount Column Volume Time in Step Mobile PhaseVolumes in mL minutes 1 Water 1.0 76.0 4.8 2 400 mM NaPi, pH 7.0 3.0228.1 14.4 3 10 mM NaPi, pH 6.8 6.0 456.2 28.8 4 10 mM NaPi, 1.0M NaCl,pH 6.5 6.0 456.2 28.8 5 Water 1.0 76.0 4.8 6 400 mM NaPi, pH 7.0 2.0152.1 9.6 7 10 mM NaPi, pH 6.8 0.5 38.0 2.4 8 Water 1.0 76.0 4.8 9 50 mMCaCl₂•2H₂O 3.0 228.1 14.4 10 Water 2.0 152.1 9.6 11 1M NaOH 2.0 152.19.6

Measurements of resin mass and particle strength were taken in threesegments of the column as in Example 3, both before the first cycle andafter the 25th cycle, and the results are shown in Table X.

TABLE X Changes in Solid Phase Mass and Strength at Three ColumnLocations Over 25 Cycles of Column Restoration Particle Strength ResinMass (UCBC @ 6.00 mm) Packing After 25 Percent Percent Location StartCycles Change N psi Change Top 25% 12.00 g 10.98 g −8.50% 142.5 537 −46%Middle 23.90 g 23.36 g −2.26% 179.1 675 −33% 50% Bottom 12.00 g 12.04 g+0.33% 209.2 788 −21% 25% Total 47.90 g 46.38 g −3.17% Control: 266.31003

Both the mass and the particle strength declined over the course of thetest, although not by as much as the control (Example 1, Table II).Nevertheless, comparison of these results with those of Example 3 (TableIV) show that improved resin restoration is achieved when the apatitesurface that has been exposed to alkali metal salt elution (Step 4) istreated with calcium ion prior to treatment with phosphate ion.

EXAMPLE 7

This example illustrates the application of the restoration method ofthe present invention to a column to which a shallow phosphate gradientsupplemented with NaCl has been applied. The column used in this examplewas 40 cm in length with an internal diameter of 1.6 cm and a flow rateof 200 cm/h or 6.70 mL/min. The packing material was the same as thatused in the preceding examples, although the packing weight was 50.67 gand the column volume was 80.42 mL. A total of 25 cycles were run, andthe protocol for each cycle was as shown in Table XI:

TABLE XI Treatment Protocol for Simulated Cycles of Separation andColumn Restoration with Phosphate Gradient Amount Column Volume Time inStep Mobile Phase Volumes in mL minutes 1 50 mM NaPi, 0.1M NaCl, pH 6.75.0 402.1 60.0 2 2 mM NaPi, 20 mM MES, 0.1M 3.0 241.3 36.0 NaCl, pH 6.73 2 mM NaPi, 20 mM MES, 10 mM 7.0 563.0 84.0 Tris, 0.1M NaCl, pH 6.7 4Water 0.08 6.4 1.o 5 20 mM NaPi, 0.1M NaCl, pH 6.7 3.0 241.3 36.0 6Gradient 8-90%: 2 mM NaPi, 10.0 804.2 120.0 0.1M NaCl, pH 6.7 → 50 mMNaPi, 0.1M NaCl, pH 6.7 7 50 mM NaPi, 0.1M NaCl, pH 6.7 2.0 160.8 24.0 850 mM CaCl₂•2H₂O 3.0 241.3 36.0 9 2 mM NaPi, 0.1M NaCl, pH 6.7 0.1 8.01.2 10 400 mM NaPi, pH 7.0 2.0 160.8 24.0 11 1M NaOH 2.0 160.8 24.0

Measurements of resin mass and particle strength are shown in Table XII.

TABLE XII Phosphate Gradient Test: Changes in Solid Phase Mass andStrength at Three Column Locations Over 25 Cycles of Column RestorationParticle Strength Resin Mass (UCBC @ 6.00 mm) Packing After 25 PercentPercent Location Start Cycles Change N psi Change Top 25% 15.02 g 15.76g +4.93% 206.0 776 −23% Middle 20.67 g 23.33 g +8.03% 214.9 810 −19% 50%Bottom 15.00 g 16.24 g +8.27% 216.6 816 −19% 25% Total 50.69 g 54.33 g+7.18% Control: 266.5 1004

While the particle strength values in Table XII show declines in allthree sections of the column, a pilot-scale column packed to 40 cm inheight and 20 cm in width was run through the same cycle withoutrestoration steps 8 and 10 and without the intermediate wash step 9(i.e., using cycles that instead consisted only of steps 1, 2, 3, 4, 5,6, 7, and 11 of Table XI). The declines in particle strength for thesecurtailed cycles in the top, middle, and bottom sections of the column,were −26%, −40% and −46%, respectively, after only eleven cycles. Acomparison between these values and those of Table XII shows thatparticle strengths in all three sections of the column declined to amuch lesser degree (and over twice the number of cycles) when therestoration steps were included in each cycle than when the restorationsteps were omitted.

In further tests, the protocol was repeated with the CaCl₂ concentrationin Step 8 was reduced to 25 mM and 2 mM, respectively, all other stepsbeing unchanged, to assess the effects of concentration on the packedresin. The results are shown in Table XIII, indicating some dependenceon the concentration of CaCl₂ with the higher concentration providing agreater degree of restoration, particularly in the upper portions of thecolumn.

TABLE XIII Phosphate Gradient Test with Variable CaCl₂ Concentration:Changes in Solid Phase Mass and Strength at Three Column Locations Over25 Cycles of Column Restoration ParticleStrength Resin Mass (UCBC @ 6.00mm) Packing After 25 Percent Percent Location Start Cycles Change N PsiChange Using 25 mM CaCl₂: Top 25% 16.66 g 172.5 650 −35% Middle 50%23.21 g 210.7 794 −21% Bottom 25% 13.41 g 198.85 749 −25% Total 50.67 g53.28 +5.15% Using 2 mM CaCl₂: Top 25% 6.57 g 152.45 574 −43% Middle 50%11.97 g 168.6 635 −37% Bottom 25% 5.49 g 194.85 734 −27% Total 23.95 g24.03 g +0.33%

Despite the loss in particle strength, the degree of loss shown in TableXIII at both concentrations is still significantly less than thatobserved in the comparison test cited above (i.e., the test using theprotocol of Table XI minus steps 8, 9, and 10 for each cycle), at leastin the middle and bottom sections of the column.

In the claims appended hereto, the term “a” or “an” is intended to mean“one or more.” The term “comprise” and variations thereof such as“comprises” and “comprising,” when preceding the recitation of a step oran element, are intended to mean that the addition of further steps orelements is optional and not excluded. All patents, patent applications,and other published reference materials cited in this specification arehereby incorporated herein by reference in their entirety. Anydiscrepancy between any reference material cited herein or any prior artin general and an explicit teaching of this specification is intended tobe resolved in favor of the teaching in this specification. Thisincludes any discrepancy between an art-understood definition of a wordor phrase and a definition explicitly provided in this specification ofthe same word or phrase.

What is claimed is:
 1. A method for at least partially reversingdeterioration of an apatite-based chromatography resin resulting frompurification of a target molecule with said resin, said methodcomprising: (a) passing a solution of calcium ion through said resin;(b) passing a solution of phosphate ion having a pH of at least about6.5 through said resin; and (c) passing a solution of hydroxide ionthrough said resin; steps (a), (b), and (c) being performed separatelyfrom any passage of equilibration, sample loading, and elution buffersthrough said resin and at concentrations of said ions and at volumes ofsaid solutions that are effective to at least partially reversedeterioration of said resin caused by said elution buffer.
 2. The methodof claim 1, wherein step (a) is performed before step (b), and step (b)is performed before step (c).
 3. The method of claim 1, wherein targetmolecules are extracted from a plurality of samples contacted insuccession with said resin, and steps (a), (b), and (c) are performedafter each of said samples is contacted with said resin.
 4. The methodof claim 1, further comprising passing an aqueous wash solution throughsaid resin after each of steps (a), and (b).
 5. The method of claim 3,further comprising passing an aqueous wash solution through said resinafter each of steps (a), (b), and (c).
 6. The method of claim 1, whereinsaid apatite-based chromatography resin is ceramic hydroxyapatite. 7.The method of claim 1, wherein said apatite-based chromatography resinis a member selected from the group consisting of fluoroapatite andfluoride-enhanced apatite.
 8. The method of claim 1, wherein saidsolution of calcium ion has a calcium ion concentration of from about 10ppm to about 2000 ppm, and is passed through said resin in an amount offrom about 1.0 resin volume to about 10.0 resin volumes.
 9. The methodof claim 1, wherein said solution of calcium ion has a calcium ionconcentration of from about 30 ppm to about 1000 ppm, and is passedthrough said resin in an amount of from about 1.5 resin volumes to about6.0 resin volumes.
 10. The method of claim 1, wherein said solution ofphosphate ion has a pH of from about 6.5 to about 9.0, has a phosphateion concentration of from about 50 mM to about 1 M, and is passedthrough said resin in an amount of from about 1.0 resin volume to about20.0 resin volumes.
 11. The method of claim 1, wherein said solution ofphosphate ion has a pH of from about 6.5 to about 7.5, has a phosphateion concentration of from about 200 mM to about 750 mM, and is passedthrough said resin in an amount of from about 1.5 resin volumes to about10.0 resin volumes.
 12. The method of claim 1, wherein said solution ofhydroxide ion has a hydroxide ion concentration of from about 0.1 M toabout 5.0 M, and is passed through said resin in an amount of from about1.0 resin volume to about 20.0 resin volumes.
 13. The method of claim 1,wherein said solution of hydroxide ion has a hydroxide ion concentrationof from about 0.3 M to about 3.0 M, and is passed through said resin inan amount of from about 1.5 resin volume to about 10.0 resin volumes.14. The method of claim 1, wherein said resin is one that has beenexposed to said elution buffer at a pH of about 8.0 or below.
 15. Themethod of claim 1, wherein said resin is one that has been exposed tosaid elution buffer at a pH of from about 6.0 to about 8.0.
 16. Themethod of claim 1, wherein said resin is one that has been exposed tosaid elution buffer containing an alkali metal halide at a concentrationof at least about 30 mM.
 17. The method of claim 1, wherein said resinis one that has been exposed to said elution buffer containing an alkalimetal halide at a concentration of from about 30 mM to about 3000 mM.18. The method of claim 1, wherein said target molecule is apolypeptide.
 19. In a method for purifying target molecules from aplurality of samples on an apatite-based chromatography resin by loadingsaid samples in succession onto said resin and eluting said targetmolecules from said resin after each such loading with an elution buffercomprising an alkali metal salt, the improvement comprising treatingsaid resin after each target molecule elution and before each subsequentsample loading by: (a) passing a solution of calcium ion through saidresin; (b) passing a solution of phosphate ion having a pH of at leastabout 6.5 through said resin; and (c) passing a solution of hydroxideion through said resin; steps (a), (b), and (c) being performedseparately from all passages of column equilibration, sample loading,and elution buffers through said resin and at concentrations of saidions and at volumes of said solutions that are effective to at leastpartially reverse deterioration of said resin caused by said elutionbuffer.
 20. The method of claim 19, wherein said plurality of samplesconsists of at least twenty samples.
 21. The method of claim 19, whereinsaid plurality of samples consists of at least fifty samples.
 22. Themethod of claim 19, wherein step (a) is performed before step (b), andstep (b) is performed before step (c).
 23. The method of claim 19,further comprising passing an aqueous wash solution through said resinafter each of steps (a), (b), and (c).
 24. The method of claim 19,wherein said apatite-based chromatography resin is ceramichydroxyapatite.
 25. The method of claim 19, wherein said apatite-basedchromatography resin is a member selected from the group consisting offluoroapatite and fluoride-enhanced apatite.
 26. The method of claim 19,wherein said solution of calcium ion has a calcium ion concentration offrom about 10 ppm to about 2000 ppm, and is passed through said resin inan amount of from about 1.0 resin volume to about 10.0 resin volumes.27. The method of claim 19, wherein said solution of calcium ion has acalcium ion concentration of from about 30 ppm to about 1000 ppm, and ispassed through said resin in an amount of from about 1.5 resin volumesto about 6.0 resin volumes.
 28. The method of claim 19, wherein saidsolution of phosphate ion has a pH of from about 6.5 to about 9.0, has aphosphate ion concentration of from about 50 mM to about 1 M, and ispassed through said resin in an amount of from about 1.0 resin volume toabout 20.0 resin volumes.
 29. The method of claim 19, wherein saidsolution of phosphate ion has a pH of from about 6.5 to about 7.5, has aphosphate ion concentration of from about 200 mM to about 750 mM, and ispassed through said resin in an amount of from about 1.5 resin volumesto about 10.0 resin volumes.
 30. The method of claim 19, wherein saidsolution of hydroxide ion has a hydroxide ion concentration of fromabout 0.1 M to about 5.0 M, and is passed through said resin in anamount of from about 1.0 resin volume to about 20.0 resin volumes. 31.The method of claim 19, wherein said solution of hydroxide ion has ahydroxide ion concentration of from about 0.3 M to about 3.0 M, and ispassed through said resin in an amount of from about 1.5 resin volume toabout 10.0 resin volumes.
 32. The method of claim 19, wherein saidelution buffer has a pH of about 8.0 or below.
 33. The method of claim19, wherein said elution buffer has a pH of about 6.0 to about 8.0.